Directed evolution of a thermostable esterase

We have used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase. Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtilis p-nitrobenzyl esterase significantly (>14°C increase in Tm) without compromising its catalytic activity at lower temperatures. Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated. Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important. Mutations that increase thermostability while maintaining low-temperature activity are very rare. Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.

Adaptation of the bovine spongiform encephalopathy agent to primates and comparison with Creutzfeldt– Jakob disease: Implications for human health

There is substantial scientific evidence to support the notion that bovine spongiform encephalopathy (BSE) has contaminated human beings, causing variant Creutzfeldt–Jakob disease (vCJD). This disease has raised concerns about the possibility of an iatrogenic secondary transmission to humans, because the biological properties of the primate-adapted BSE agent are unknown. We show that (i) BSE can be transmitted from primate to primate by intravenous route in 25 months, and (ii) an iatrogenic transmission of vCJD to humans could be readily recognized pathologically, whether it occurs by the central or peripheral route. Strain typing in mice demonstrates that the BSE agent adapts to macaques in the same way as it does to humans and confirms that the BSE agent is responsible for vCJD not only in the United Kingdom but also in France. The agent responsible for French iatrogenic growth hormone-linked CJD taken as a control is very different from vCJD but is similar to that found in one case of sporadic CJD and one sheep scrapie isolate. These data will be key in identifying the origin of human cases of prion disease, including accidental vCJD transmission, and could provide bases for vCJD risk assessment.

Enzymatic phosphorylation of muscle glycogen synthase: a mechanism for maintenance of metabolic homeostasis.

We recently analyzed experimental studies of mammalian muscle glycogen synthesis using metabolic control analysis and concluded that glycogen synthase (GSase) does not control the glycogenic flux but rather adapts to the flux which is controlled bv the activity of the proximal glucose transport and hexokinase steps. This model did not provide a role for the well established relationship between GSase fractional activity, determined by covalent phosphorylation, and the rate of glycogen synthesis. Here we propose that the phosphorylation of GSase, which alters the sensitivity to allosteric activation by glucose 6-phosphate (G6P), is a mechanism for controlling the concentration of G6P instead of controlling the flux. When the muscle cell is exposed to conditions which favor glycogen synthesis such as high plasma insulin and glucose concentrations the fractional activity of GSase is increased in coordination with increases in the activity of glucose transport and hexokinase. This increase in GSase fractional activity helps to maintain G6P homeostasis by reducing the G6P concentration required to activate GSase allosterically to match the flux determined by the proximal reactions. This role for covalent phosphorylation also provides a novel solution to the Kacser and Acarenza paradigm which requires coordinated activity changes of the enzymes proximal and distal to a shared intermediate, to avoid unwanted flux changes.